The important role for the renal adenosine 5'-triphosphate sensitive potassium (K/ATP) channel ROMK in the kidney was verified by the demonstration that mutations in the human ROMK gene result in Bartter's syndrome, a salt wasting disorder. Hence, processes that modulated ROMK function influence K+ homeostasis and salt absorption. Although it is known that intracellular ATP regulates K/ATP (including ROMK) channel activity, there is little knowledge about the molecular nature of this regulation or the factor(s) which may modulate it. This research project will investigate the characteristics of nucleotide binding to the renal K/ATP channel ROMK, and how internal pH and the phosphorylation state of the channel protein modulate this binding itself. The aims of the proposal are: 1) Molecular characterization of the nucleotide binding to the C-terminus region of ROMK; 2) Modulation of nucleotide binding to the C-terminus region of ROMK by pH and the phosphorylation state; 3) role of ROMK as an ATPase; and, if time allows, 4) Characterization of nucleotide binding to the C-terminal domain of the K/ATP channel Kir6.2 The data obtained will provide valuable information for understanding the processes by which nucleotides regulate ROMK and other K/ATP channels and provide insights into the physiology of K/ATP channels. Several methods will be used to meet these goals including molecular biology techniques (e.g., site-directed mutagenesis) protein biochemistry techniques (e.g., protein isolation and purification), electrophysiological techniques (2- electrode voltage-clamp), and quantitative fluorescent techniques.